Normally, the hemoglobin in circulating blood is usually more than 99% in the ferrous (Fe.sup.2+) form. The ferrous form transports oxygen. Another form of hemoglobin, known as methemoglobin, contains the ferric ion (Fe.sup.3+). Methemoglobin is unable to transport oxygen. In healthy individuals, the methemoglobin concentration in circulating blood is usually less than one percent. The iron in hemoglobin is kept in the ferrous valence state by the activity of a special methemoglobin reductase system. See Metzler, Biochemistry, Academic Press, New York, N.Y., 1997, pp. 564-565.
Methemoglobin is monitored to evaluate biological exposure to biotoxins and possible carcinogenic compounds which contain primary aromatic amino-, nitro-, nitroso-, azo- and/or certain phenolic moieties. These residues have been directly correlated to methemoglobin production both in vivo and in vitro. An elevated methemoglobin concentration can be indicative of exposure to these and/or other toxicants.
The normal methemoglobin concentration is less than 1% in normal, living persons. Shortly after blood is drawn from a living person, or after a person dies, the methemoglobin enzymatic reduction systems become disrupted and the hemoglobin iron starts moving towards a new equilibrium between the ferrous and the ferric state, i.e., from normal Fe.sup.2+ hemoglobin toward Fe.sup.3+ methemoglobin. In less than one hour after blood collection, the methemoglobin concentration can become spontaneously elevated to a level which is above the biological exposure limit of 1.5%, resulting in an erroneous interpretation of excessive toxicant exposure. In forensic cases, spontaneous bacterial or other thanatologic causes can not only produce apparently "toxic" levels of methemoglobin, but can also result in lowering causally significant levels of methemoglobin, thus rendering those levels misinterpretable.
The ability to accurately measure methemoglobin is presently limited to a narrow time window after sample collection via venipuncture or other collection methods, due to the spontaneous oxidation of hemoglobin to methemoglobin. What is needed is a quantitative methemoglobin analysis method wherein the time from blood collection to analysis may be extended without the risk of spontaneous methemoglobin production in the blood sample, which would otherwise undesirably increase the actual methemoglobin concentration present at the time of collection.